3.2 Site-specific PEGylation of FGF2 variants
We used 5kDa monomethoxy PEG-maleimide (mPEG-MAL) to specifically modify the surface-exposed cysteine on FGF2 and the mutants, as shown inFigure 2A . Three mono-PEGylated FGF2 conjugates were generated and named as PEG-FGF2C69A/C87A/F17C (compound5 ), PEG-FGF2C87A (compound 6 ), and PEG-FGF2C69A/C87A/K129C (compound 7 ) (Figure 2B) . SDS-PAGE analysis confirmed the purification of these PEGylated FGF2 conjugates. A single band corresponding to protein with an apparent Mw of 25kDa appeared after PEGylation (Figure 2C ). After purification, the ERK1⁄2 pathway activity was measured. All PEG-FGF2 conjugates and native FGF2 exhibited higher ERK1/2 phosphorylation activity than that of the PBS control. In addition, compared with FGF2, compound 5 and 6 induced about 1.5- and 2- fold higher phosphorylation, and compound 7exhibited a significant reduction in phosphorylation (Figure 2 D-E ). The decreased activity of compound 7 , in which PEGylation would occur at the engineered cysteine near the heparin-binding region, suggests that steric hindrance of the PEG polymers can interfere with binding between heparin and the receptor. The observed sensitivity of the heparin-binding region suggests designs that do not interfere with this region of the protein may result in FGF2 conjugates with increased activity retention and stability after site-specific PEGylation.